Method of destroying bacteria employing mercuric-chloride complexes of pyridine



Patented Oct. 8, 1957 METHOD OF DESTROYING BACTERIA EMPLOY- INGMERCURIC-CHLORIDE COMPLEXES F PYRIDINE Charles W. Osborn, Bartlesville,0kla., and Raymond C. Allred, Kirkwood, Mo., assiguors to PhillipsPetroleum Company, a corporation of Delaware No Drawing. ApplicationSeptember 21, 1954, Serial No. 457,564

9 Claims. (Cl. 167-33) This invention relates to novel bactericidalcompounds, compositions and methods of applying the same. In one of itsaspects, the invention provides a novel use for mercuric chloridecomplexes of esters of pyridine-2,5- dicarboxylic acid. In another ofits aspects, the invention provides a novel method for the control ofbacteria which comprises applying to said bacteria a mercuric chloridecomplex of an ester of pyridine-2,5-dicarboxylic acid. In a specificaspect, the invention provides as novel compositions, havingbactericidal properties, mercuric chloride complexes of diethylanddi-n-propyl esters of pyridine- 2,5-dicarboxylic acid dispersed in abactericide adjuvant as a carrier therefor. Other aspects as well asadvantages of the invention are apparent from this disclosure and theappended claims.

The mercuric chloride complexes of esters of pyridine- 2,5-dicarboxylicacid are prepared by bringing together mercuric chloride, dissolved in,say, absolute ethyl alcohol, and the esters of said acid. In a specificpreparation, the modus operandi was as follows:

EXAMPLE I Mercuric chloride complex of the diethyl ester ofpyridine-2,5-dicarboxylic acid was prepared in the following manner. 6.8grams, 0.025 mols, of HgCl2 was dissolved by heating in 20 mls. ofabsolute ethyl alcohol. An equal molar quantity, 0.025 mol, or 5.58grams of the diethyl ester of pyridine-2,5-dicarboxylic acid M. P. 46-50 C. was dissolved in 10 mls. of absolute ethyl alcohol and added tothe previously prepared mercuric chloride solution. Crystals formedrapidly, and the entire contents set up into a solid mass. This productwas cooled and an additional 10 mls. of absolute ethyl alcohol wasadded. The product was then filtered, washed with 20 mls. absolute ethylalcohol, and dried overnight at 70 C. The mercuric chloride complex ofthe diethyl ester of pyridine- 2,5-dicarboxylic acid which was obtainedwas found to be 10.4 grams with a M. P. of 132 434 C., i. e., a yield of84 percent.

In another specific preparation, the details were as follows:

EXAMPLE II Mercuric chloride complex of the di-n-propyl ester ofpyridine-2,S-dicarboxylic acid was prepared in the following manner. 7.1grams of HgClzwas dissolved in 30 mls. absolute alcohol and to this wasadded 6.6 grams of the di-n-propyl ester of pyridine-2,5-dicarboxylicacid. It was not necessary to dissolve the ester in this case since itwas already liquid. The solution was then heated to boiling, cooled inan ice chest, and seeded to induce crystallization. The crystals thatwere formed were then filtered and washed with 30 mls. of cold absoluteethyl alcohol. The yield of the mercuric chloride complex of thedi-n-propyl ester of pyridine-2,S-dicarboxylic acid was found to be 10.2grams representing a yield of percent. The product was recrystallizedfrom absolute ethyl alcohol and its M. P. was found to be 82 C.

The n-propyl complexes were titrated with perchloric acid in glacialacetic acid to determine the molar ratio of salt to ester and it wasfound to be 1:1.

Calculated: HgCl2.C13HnO4N, 523. Found, 520.

The complexes of the invention can be represented by the general formulaor molecular arrangement as follows:

R-OOC When R is C2H5 and/or n-CsH7 the compounds are the diethyl-, thedi-n-propyl-, or the mixed ethyl-npropyl esters ofpyridine-2,S-dicarboxylic acid.

The following are physical properties of the diethyl and di-n-propylcomplexes. The mercuric chloride complex of the di-n-propyl ester ofpyridine-2,S-dicarboxylic acid has a M. P. of 8082 C., is soluble inacetone, methyl alcohol, ethyl acetate, and ethyl alcohol, and isinsoluble in water and chloroform. The mercuric chloride complex of thediethyl ester of pyridine-2,5-dicarboxylic acid has a M. P. of 132-134C., is soluble in acetone, ethyl acetate, hot methyl alcohol, ethylalcohol, and hot water, and is insoluble in cold water.

While in the bactericidal method of the invention, the last two namedcompounds are preferred, the R can include up to six carbon atoms ineach alkyl group.

It is preferred to use these new bactericides dissolved in a bactericideadjuvant, for example, ethyl alcohol, preferably in concentrations of0.01 percent to 1 percent bactericide by Weight. However, weaker orstronger concentrations may be used depending on the degree ofbactericidal effectiveness desired, and the total quantity ofbactericidal solution applied. The preferred method of controllingbacteria according to the invention is to directly contact the bacteriawith a solution as here described. As other satisfactory bactericideadjuvants there can be used water, mixtures of water and alcohol, andacetone, either alone or in admixture with alcohol or water or both.

EXAMPLE III ment, Chemical Corps, Camp Detrick, Maryland. The

procedure is as follows:

Several glass Petri dishes were sterilized and provided with Breweraluminum tops. These aluminum tops had paper disk centers to keepmoisture from collecting above the dishes and dripping down therefrom.Into the glass dishes was pipetted 15 cos. of sterile penicillin assaybase agar, consisting of 6 grams peptone, 3 grams yeast extract, 1.5grams beef extract, and 15 grams of agar in 1000 cos. of Water. Whenthis agar had hardened 5 cos. of inoculated penicillin. assay seed agarwas added. This seed agar was composed of 6 grams peptone, 4 gramstrypticase, 3 grams yeast extract, 1.5 grams beef extract,

3 1 gram glucose, and 15 grams of agar in 1000 ccs. of Water, andhadbeen inoculatedby adding 0.3 cc. of 48 hour broth culture of eitherMicrococcus pyogenes var.

4 The same test for bactericidal properties was run on commercialtincture of merthiolate and solutions of commercial mercuric chloride.The results of these tests are aureus or Escherichia coli to each 100ccs. of agar. given below.

When the media had hardened, a standard penicillin Table III assay disk,a thick porous paper 12.5 mms. in diameter, was placed in the center ofeach plate. A solution of either Zone of Inhibimn the mercuricdl-n-propyl ester of pyridine 2,5-d1car- Goncentra- After 48 hours(mrn.) boxylic acid or the mercuric chloride complex of the di- Compoundf g h ethyl ester of pyridine 2,5-dicarboxylic acid in absolute 1S.aureus E. coli ethyl alcohol was placed on the disk in the amount of()7 llt ierthiolag iu d 1,000 33.5 20 The plates were then incubated at37 C. and read at 24 83. 2: 388 g; and 48 hours. The zone of inhibitionwas read by holding the plate to the light and measuring the generallycir- Tabl IV cular zone where no bacteria growth occurred. A zone 8 of12.5 mms. indicates no inhibition since it is the diameter (Comparative)of the test disk used. The results of these runs are tabulated below. Ct Az ig h z 011C811 ra- Cl OllIS mm. Table I Compound tion, HgOl COMPLEXOF DIE'IHYL ESTER OF PYRIDINE 2,5-DI 2 CARBOXYLIC ACID HgClz Complex ofthe Diethylmmblt'lm (mm) ester Pyridine-2,5-Diearboxylic a i du uflu nuu 1, 000 33 25 g z omp ex 0 -11- ropy Concentration S. aureus E. coltEster of Pyridineajgmcap Marri i222; er ie a e .5 20 24 48 MereuricChloride 1,000 31 33 3 10,000 p. p. m 43 42 37 36 0 1,000 p. p. m... 3:g3 Z0 2? Table IV shows that these new bactericides compare E g- RE: g 2f favorably with known bactericides such as merthiolate 1 p. p. m 12.512.5 12.5 12.5 and mercurlc chloride.

T bl EXAMPLE IV a e H N T N A broth was made up containing 1.5 gramsproteose HEGIZGOMPLEX B A %%Yf i OF PYRIDI E215 peptone, 1.5 gramsneopeptone, 1.5 grams yeast extract,

and 1500 mls. of water. The pH of this broth was ad- Zone ounmbmon (mm'40 justed to 7.2, and the broth was then steam sterilized for 15 minutesat a pressure of 15 pounds gauge. A sample Concentration 5.1mm E10 ofthis broth was then inoculated with one drop of an actively growing 24hour culture of the organism on 24 hr 48 hr 24 hr 48 hr. which thebactericide was to be tested. Four ml. samples of this inoculated brothwere placed in 13 by 100 mm. 10,000 41 40 31 30 test tubes, anddesignated amounts of the bactericides r3500 p. p. m-.. 2% were added togive the desired concentration of bacteri- 10 35 1 3 16 15 15 14 cide.These samples were then incubated at 25 C. and 1p.p.m 15 12,5 12.5 12.5the results read at 24 and'48 hours. The concentration of bactericide isbased on 100 percent active ingredients.

Table V TESTING OF H ch COMPLEX OF THE DI-N-PROPYL ESTER 0F PYRIDINE-2,5-DIOARBOXYLIO ACID AGAINST VARIOUS ORGANISMS Cntrolo Organism 1p.p.m.5p.p.'m. 10 25 50 Time (No Bacp. p. m. p. p. m. p. p. m. p. p. in. hourstericide Present) :t: i 24 Escherzchta colt i d: ii Pseudomonaa speczeii i g; :2 ii

. d: BGCZZZ'LLS subttha ii d: d: d: ii Serratza marcescens t: 3: l 32 iiProteus vulgam g i i i g2 ii Klebszella pneumomae. i d: 22 ii Aerobacteraerogenes d: d: d: 32 ii Staphylcaccus omens"... d: Sarcma apecze iiBacillus cereus 48 Full growth, identical with control.

Table VI TESTING OF HgClQ COMPLEX OF THE DIETHYL ESTER OF PYRIDINE-2,5-DICARBOXYLIC ACID AGAINST VARIOUS ORGANISMS Cone. of BaetericidePresent Control Time (No Bao- Organlsm hours tericide 1p.p.m. p.p.m. 50100 Present) p. p. m p. p m. p. p. m. p. p. m.

Escherichia coli j; I I I I I 2% Pseudomomzs specie I I I i I I Bacillusaubtilis i i I Z Serratz'a martescem I; I I I I 2; Proteus vulaaria i II I I I 2% Klebsiella pneumoniae. i 3 I I I I f; Aerobacter aeroamea i ii I 2; Staphulcoccus auntie--." I I I l I I I g Sarcina specie"; I I I II I 23 I Bacillus cereus i I I I I I ii ++=Fu1l growth. identical withcontrol.

+=Fair growth. =I==Little growth. =No growth.

Mercuric chloride complexes of esters of pyridine-2,5- dicarboxylic acidand a method for preparation of same are disclosed and claimed in thecopending application of Carl W. Kruse and A. Merrill Schnitzer, SerialNo. 457,568, filed even date September 21, 1954.

l'xeasona'ole variation and some modification are pos sible within thescope of the foregoing disclosure and the appended claims to theinvention, the essence of which is that certain mercuric chloride estersof pyridine-2,5- dicarboxylic acid, especially the diethyland thedi-npropyl esters, have been set forth as bactericidal in character, amethod for controlling or killing bacteria employing at least one ofsaid compounds has been set forth and that a novel bactericidalcomposition has been, set forth all substantially as described.

We claim:

1. A method of inhibiting the growth of bacteria which comprisescontacting said bacteria with a mercuric chloride complex of an ester ofpyridine-2,S-dicarboxylic acid in an amount eifective to inhibit thegrowth of said bacteria.

2. A method of inhibiting the growth of bacteria which comprisescontacting said bacteria with a mercuric chloride complex of diethylester of pyridine-2,5-dicarboxylic acid in an amount effective toinhibit the growth of said bacteria.

3. A method of inhibiting the growth of bacteria which comprisescontacting said bacteria with a mercuric chloride complex of di-n-propylester of pyridine-2,5-dicarboxylic acid in an amount effective toinhibit the growth of said bacteria.

4. A method of preventing the growth of bacteria which comprisesapplying to said bacteria a solution of a mercuric chloride complex ofan ester of pyridine-2,5- dicarboxylic acid dissolved in a bactericideadjuvant.

5. A method according to claim 4 wherein the adjuvant is a materialselected from the group of solvents consisting of ethyl alcohol,acetone, a mixture of water and alcohol, a mixture of acetone andalcohol, and a mixture of acetone, alcohol and water.

6. A method according to claim 5 wherein the complex is a complexselected from the group consisting of diethyland di-n-propyl esters ofpyridine-2,5-dicarboxylic acid.

7. A method of controlling bacteria which comprises applying to saidbacteria CaHs-OOC 8. A method of controlling bacteria which comprisesapplying to said bacteria nCaHr-O 00 9. A method of controlling bacteriawhich comprises applying to said bacteria OTHER REFERENCES Frear: Chem.of Insecticides, Fungicides and Herbicides, 2nd ed. (1948), p. 254.

1. A METHOD OF INHIBITING THE GROWTH OF BACTERIA WHICH COMPRISESCONTACTING SAID BACTERIA WITH A MERCURIC CHLORIDE COMPLEX OF AN ESTER OFPYRIDINE-2,5-DICARBOXYLIC ACID IN AN AMOUNT EFFECTIVE TO INHIBIT THEGROWTH OF SAID BACTERIA.